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1 HEMATOLOGIC EFFICACY OF PEGYLATED FELINE GRANULOCYTE COLONY STIMULATING FACTOR (G CSF) IN FIV INFECTED AND UNINFECTED CATS By YOHICHI SAKAGAWA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PART IAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011
2 2011 Yohichi Sakagawa
3 I would like to thank my parents for all their unconditional love, support, guidance and discussion throughout the year s of undergraduate and graduate degree. I am also dedicating this work to my cat, May, who passed away from a FIV infection during my last year as an undergraduate student. Lastly, I dedicate this work to the animals that devoted their precious life for my research.
4 ACKNOWLEDGMENTS I would like to express special thanks to my mentor, Dr. Janet K. Yamamoto, Ph.D., for her genuine concern regarding my future and her constant mentorship throughout my attendance at the College of Veterinary Medicine, Uni versity of Florida. If not for her, I would never have had the opportunity to work on this wonderful and exciting project. I would also like to express my gratitude to her for offering a tremendous amount of help on the final editing of this work. I ext end special thanks to Dr. Ruiyu Pu, who helped me with all of the animal work and in vitro assays related to my project, and Dr. James K. Coleman for his involvement in the production of FeG CSF and PegFeG CSF his assistance with the NFS 60 proliferation a nd neutralization assays and with the molecular techniques used in this project. I would also like to thank the lab technicians for helping with my animal work. Lastly, I would like to convey my appreciation to the members of my supervisory committee (Dr Charles H. Cou r tney, DVM, Ph.D. and Dr. Rowan J. Milner, DVM, Ph.D.) for their knowledge and support with this project. Part of our work was supported by Harold H. Morris Trust Fund for Research in the Field of Diseases of Small Animals and by Dr. Yamam oto Miscellaneous Donors Fund.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ............................. 9 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 BACKGROUND ................................ ................................ ................................ ...... 13 2 REVIEW OF LITERATURE ................................ ................................ .................... 16 2.1 G CSF Structure and G CSF Receptor ................................ ......................... 16 2 .2 G CSF on Hematopoietic and Non hematopoietic Cells ............................... 18 2.3 Intracellular Signaling from G CSF Receptor Ligation ................................ ... 21 2.4 Interaction with Other Cytokines ................................ ................................ ... 22 2.5 HuG CSF and Species specific G CSF ................................ ........................ 23 2.6 Pegylation ................................ ................................ ................................ ..... 25 3 MATERIALS AND METHODS ................................ ................................ ................ 27 3.1 Animals ................................ ................................ ................................ ......... 27 3.2 Production of FeG CSF and PegFeG CSF ................................ ................... 28 3.3 Treatment Dose and Schedule ................................ ................................ ...... 28 3.4 Hematological Values ................................ ................................ ................... 29 3.5 Neutralizing Antibodies ................................ ................................ ................. 29 3.6 Pilot Study of PegFeG CSF Application in Dogs ................................ ........... 30 3.7 Statistical Analysis ................................ ................................ ........................ 30 4 RESULTS ................................ ................................ ................................ ............... 35 4.1 Comparison of Neutrophil Production between FeG CSF and PegFeG CSF ................................ ................................ ................................ ..................... 35 4.2 Major Difference in Neutrophil Produc tion between PegFeG CSF and HuG CSF ................................ ................................ ................................ ............. 35 4.3 Additional HuG CSF Study to Determine the Duration of HuG CSF Induced Neutropenia ................................ ................................ ........................... 37 4.4 Effect of Age and FIV Status on Neutrophil Levels of HuG CSF or PegFeG CSF Treated Cats ................................ ................................ ................. 38 4.5 Determining the Optimal Blood Collection Schedule ................................ ..... 39
6 4.6 Neutralizing Antibodies to HuG CSF and PegFeG CSF ............................... 40 4.7 Other Hematological Values, Clinical Signs, and FIV Load ........................... 40 4.8 PegFeG CSF Therapy of HuG CSF Induced Neutropenia ........................... 41 4.9 Inter species Use of PegFeG CSF ................................ ................................ 42 5 DISCUSSION ................................ ................................ ................................ ......... 53 LIST OF REFERENCES ................................ ................................ ............................... 57 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 67
7 LIST OF TABLES Table page 3 1 Brief description of animals used in this study ................................ .................... 32 3 2 Statistical analysis ................................ ................................ .............................. 33
8 LIST OF FIGURES Figure page 3 1 SDS PAGE with silver stain for purification of PegFeG CSF .............................. 34 4 1 Neutrophil numbers in cats treated with FeG CSF (Fe) or PegFeG CSF (Peg) in Cycle 1 ................................ ................................ ................................ .. 44 4 2 Decreased numbers of neutrophils due to anti drug neutralizing antibodies and activities were seen in cats treated with HuG CSF (Panel A) but not in PegFeG CSF tr eated cats (Panels A and B) ................................ ...................... 45 4 3 Comparison of hematological blood smears from pre and post treatment samples ................................ ................................ ................................ .............. 47 4 4 Addi tional HuG CSF study to characterize HuG CSF induced neutropenia ....... 48 4 5 PegFeG CSF therapy of cats with HuG CSF induced neutropenia .................... 49 4 6 Effect of age and FIV infection on neutrophil levels among cats treated with HuG CSF or PegFeG CSF ................................ ................................ ................. 50 4 7 Comparison of G CSF sequences between Hu Fe and Ca G CSF ............... 51 4 8 Inter species use of PegFeG CSF ................................ ................................ ...... 52
9 LIST OF ABBREVIATION S BLAST Basic Local Alignment Search Tool CaG CSF Canine G CSF CHO Chinese hamster ovary EPO Erythropoie tin FeG CSF Feline G CSF FIV Feline immunodeficiency virus G CSF Granulocyte colony stimulating factor G CSFR G CSF receptor GM CSF Granulocyte/macrophage colony stimulating factor GVDH Graft versus host disease HuG CSF Human G CSF IACUC Institutional An imal Care and Use Committee IFN Interferon IL Interleukin JAK Janus kinase LCIR Laboratory of Comparative Immunology and Retrovirology LPS Lipopolysaccharide MAPK Mitogen activated protein kinase M CSF Monocyte colony stimulating factor PBS Phosphate buff ered saline PEG Polyethylene glycol PegFeG CSF Pegylated FeG CSF PegHuG CSF Pegylated HuG CSF PI 3K Phospho inositol 3 kinase
10 PKB Protein kinase B SDS PAGE S odium dodecyl sulfate polyacrylamide gel electrophoresis SHIP SH2 containing inositol phosphatase S OCS S uppressor of cytokine signaling SPF Specific pathogen free STAT Signal Transducers and Activators of Transcription Th T helper TNF Tumor necrosis factor
11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science HEMATOLOGIC EFFICACY OF PEGYLATED FELINE GRANULOCYTE COLONY STIMULATING FACTOR (G CSF) IN FIV INFECTED AND UNINFECTED CATS By Yohichi Sakagawa December 2011 Chair: Janet K Ya mamoto Major: Veterinary Medical Science s Neutropenia is a common hematologic problem in patients with hematopoietic disorders and immunodeficiency syndromes, which can often be corrected by the treatment of granulocyte colony stimulating factor (G CSF). Off label use of commercial human G CSF (HuG CSF) has become a common procedure for treatment of neutropenia in cats and dogs. However, long term treatment with HuG CSF causes adverse effects that include loss of drug activity and neutropenia. In the cu rrent stud y, pegylated recombinant feline G CSF (PegFeG CSF) was produced and tested in cats. A randomized controlled clinical trial was conducted to evaluate the hematological efficacy of PegFeG CSF compared to either FeG CSF or HuG CSF (Filgrastim ) in FIV infected (n=10) and uninfected (n= 9 ) healthy cats, and in cats with HuG CSF induced neutropenia. Weekly doses of PegFeG CSF induced higher peak neutrophil production and showed greater sustained activity than weekly treatment with a similar dose of Fe G CSF ( p =0.002) or daily HuG CSF treatments ( p =0.018). PegFeG CSF provided the most therapeutic and sustainable neutrophil production ( p <0.001) in both uninfected and FIV infected cats without the development of neutralizing antibodies to the drug. In co ntrast, all HuG CSF treated cats developed neutralizing antibodies to HuG CSF,
12 suggesting the presence of cross reactive antibodies to endogenous G CSF in a majority of the cases with severe neutropenia. A striking observation was the therapeutic efficacy of PegFeG CSF rescuing animals with HuG CSF induced neutropenia resulting in return of clinically normal neutrophil numbers ( p =0.039). Thus, PegFeG CSF appears to be a superior treatment for neutropenia in feline patients
13 CHAPTER 1 BACKGROUND Neutropen ia, a common problem in patients with hematopoietic disorders and immunodeficiency syndromes, is frequently treated with the hematological growth factor, granulocyte colony stimulating factor (G CSF) (Ogilvie, 1995). Both endogenous and recombinant G CSFs exert their therapeutic effect by enhancing the development and differentiation of neutrophils from myeloid progenitor cells (Beekman and Touw, 2010). Human G CSF (HuG CSF; Filgrastim Amgen Inc., Thousand Oaks, CA) (Amgen Inc., 2004) is currently used a s therapy in both humans and animals (Fernndez Varn et al., 2007). However, continuous use of this human derived product in animals is reported to induce neutralizing antibodies against the drug and potentially against the endogenous protein (Arai et al ., 2000 ; Hammond et al., 1991; MacLeod et al., 1998; Phillips et al., 2005; Randolph et al., 1999). A study using HuG CSF in clinically normal dogs further demonstrated drug induced neutralizing antibodies that can induce chronic neutropenia by inhibiting both HuG CSF and endogenous canine G C SF (CaG CSF) activities (Randolph et al., 1999). Neutralizing antibodies are thought to be produced in animals to the foreign antigenic regions of the HuG CSF (Randolph et al., 1999). Thus, species specific growth f actors, including CaG CSF and feline G CSF (FeG CSF), have been developed to avoid neutralizing antibody production, but none are commercially available (Hammond et al., 1991; Phillips et al., 2005; Randolph et al., 1999; Yamamoto et al., 2002; Yamamoto et al., 2009). Our previous studies demonstrate the benefits of treating neutropenia with recombinant FeG CSF in cats with feline immunodeficiency virus (FIV) infection ( Tanabe et al., 2004; Phillips et al., 2005). Treatment with FeG CSF increases
14 neutrophi l counts of both FIV infected and uninfected cats to higher levels than the current recommended therapy with HuG CSF (Phillips et al., 2005). Unfortunately, the short half life of both FeG CSF and HuG CSF requires daily treatments to sustain therapeutic l evels of neutrophils. In fact, the withdrawal of FeG CSF or HuG CSF treatment results in an immediate decrease in neutrophils to pre treatment levels (Phillips et al., 2005). Pegylation, the attachment of polyethylene glycol to therapeutic proteins, is an effective modification that extends the half life of the protein (Harris and Chess, 2003). This strategy has proven successful in the use of growth factors in humans (Molineux, 2003) and animals (Curran and Goa, 2002). A single injection of pegylated HuG CSF (PegHuG CSF; Pegfilgrastim, Amgen Inc., Thousand Oaks, CA) is comparable in efficacy to twice daily injections of HuG CSF in the course of chemotherapy (Lord et al., 2001). The introduction of PegHuG CSF and subsequent decrease in dosing frequenc y has been beneficial for the comfort and convenience of both the patient and physician (Molineux, 2003). For our studies recombinant FeG CSF and PegFeG CSF were produced by our laboratory and evaluated in a randomized controlled clinical trial using FIV infected and uninfected cats, and cats with HuG CSF induced neutropenia. The goals of current studies were three fold: 1) Test whether PegFeG CSF will increase neutrophil levels faster, higher, and for a more sustained period of time than FeG CSF and th e currently recommended therapy with HuG CSF. 2) Assess whether the pegylation and the use of species specific protein (i.e., PegFeG CSF) will prevent the development of
15 neutralizing antibodies. 3) Lastly, evaluate whether the cats with HuG CSF induced neutropenia will respond therapeutically to treatment with PegFeG CSF.
16 CHAPTER 2 REVIEW OF LITERATURE 2.1. G CSF Structure and G CSF Receptor G CSF is 19.8 kD glycoprotein, consisting of 173 amino acids and O linked glycosyl group at Thr133 ( Marino et al. 2001 ; Tehranchi et al., 2003) The biological activity is dependent on the presence of two disulfide bonds (Marino et al., 2001). Three dimensional structure suggests that the G CSF form is predominantly helical (104 of 175 amino acids), consisting of four alpha helix structures (Hill et al., 1993). Similarly, helical connection including two long cross over in two helixes are previously reported in the other cytokines and growth hormone granulocyte/macrophage colony stimulating factor (GM CSF), inter feron(IFN) interleukin(IL) 2, and IL 4. T hese cytokines, at least in part, share common receptor binding actions (Hart et al., 2009). Human G CSF gene is encoded in chromosome 17 and has a close relationship with the IL 6 gene (Demetri and Griffin 1991 ; Rutell a et al., 2005) Monocyte and macrophage lineage cells are the main producers of G CSF, while endothelial cells, fibroblasts, and mesothelial cells are also involved (Demetri and Griffin 1991 ; Demetri et al., 1989) Generally the level of G CSF in bloo d cir culation is consistent, which immediately increases in response to infection or cytotoxic treatment such as chemotherapy ( Koeffler et al., 1987 ; Watari et al., 1989) High levels of G CSF production are observed in various kinds of tumor cells such a s bladder carcinoma ( Welte et al 1986) hepatoma ( Gabrilove et al, 1985) squamous carcinoma ( Nagata et al, 1986) and fibrosarcoma ( Tsuchiya et al., 1986) where these cells require neutrophils to form solid colonies around them. In addition, G CSF is mob ilized upon stimulation of lipopolysaccharide (LPS). Compared to other cytokines G CSF becomes quickly active
17 since it is needed for immediate and chronic inflammatory responses ( Koeffler et al., 1988) G CSF receptor (G CSFR) is classified as a part of t he hematopoietin receptor family ( Cosman 1993, Fukunaga et al., 1990) G CSFR is a single pass trans membrane receptor consisting of 813 amino acids ( Aritomi et al., 1999) and forms a 2:2 tetrameric complex upon binding G CSF ( Fukunaga et al., 1991) The primary role of this receptor is to differentiate myeloid precursors, activate and prolong s urvival of mature neutrophils, aggregate platelets, and to translocate vascular endothelial cells ( Aaronson and Horvath, 2002) G CSFR is expressed in myeloid pro genitor cells for their proliferation and differentiation, in mature neutrophils for their mobility and survival, to induce modification of cytokine production in monocytes, in cancer cells for their own protective usage, and in normal B and T lymphocyte f or immune regulation ( Hanazono et al., 1990 ; Khwaja et al., 1993 ; Shimoda et al., 1993 ; Tsuchiya et al., 1993 ; Corcione et al., 1996 ; Morikawa et al., 1996 ; Matsushita and Arima, 1998 ; Boneberg et al., 2000 ; Morikawa et al., 2002 ; McCracken et al., 1996 ) Mature neutrophil s express the highest amount of G CSFR but the amount is still low (50 500/cell) ( Demetri and Griffin, 1991) Moreover, G CSFR is also expressed on non hematopoietic tissues especially in fetal organs and vascular endothelial cells ( C alhoun et al., 1999 ; McCracken et al., 1999 ; Crea et al., 2009) Functional G CSFR is observed on trophoblastic cells as well ( Kidd 2009) Additionally, G CSFR is expressed on brain cells, and both the anti inflammatory and anti excytotoxic effect are f avored for post stroke rehabilitation ( Zhao et al., 2007) Two membrane pr oximal regions, box 1 and box 2, are embedded in G CSFR, which are also seen in erythropoietin (EPO) receptor, IL 2 and IL 3 receptor
18 ( Murakami et al., 1991 ; Barge et al., 1996). T hese membrane proximal regions are necessary for the proliferative activity of the cells ( Hibi et al., 1990). The third membrane proximal region, box 3, is involved in differentiation of myeloid progenitors and phagocytosis of neutrophils ( Saito et al., 1 992 ; Dong et al., 1993 ; Fukunaga et al., 1993 ; Santini et al., 2003) Mutation of G CSFR is reported in some diseases such as severe congenital neutropenia ( Zavala et al., 1999) Therefore, not only does the deficiency of G CSF itself cause hematologica l abnormalities, but mutations in the receptor can also drive these abnormalities. 2.2. G CSF on Hematopoietic and Non hematopoietic Cells The primary function of G CSF is to regulate and stimulate differentiation, proliferation and growth of neutrophils f rom early myeloid lineage cells (Ogilvie, 1995). However, there have been a number of reports that demonstrate G CSF acts on the other hematopoietic lineage cells expressing G CSF R, s uch as cardiomyocytes, neuronal precursors, epithelial cells, and placen tal tissues. G CSF has been reported to show immune regulatory abilities by acting upon T cells. T cells stimulated with G CSF secrete immune soluble factors. This effect can be enhanced by further stimulation with lipopolysaccharides (LPS), thereby inh ibiting the inflammatory activity of other cytokines in the microenvironment ( Rutella 2007 ; Rutella et al., 1999 ; Hartung et al., 1995 ; Rutella et al., 1997) G CSF is also reported to directly act on T cells by switching its profile more to a T helper ( Th) 2 type phenotype ( Franzke et al., 2003). CD8+ T cells express G CSF receptor and are involved in such a profile shift ( Franzke et al., 2003). Whether CD4+ T cells express the receptor is controversial. Furthermore, the immune regulatory effect is se en in transplantations
19 where the mobilization ability of G CSF plays an important role in the prevention of graft versus host diseases (GVHD). G CSF treatment modulates the balance between Th1 and Th2 by increasing production of IL 4 and decreasing produc tion of IFN ( Sloand et al., 2007). G CSF stimulation induces the production of G CSFR and the transcriptional factor GATA 3 which then work to further promote a Th2 type profile shift ( Franzke et al., 2003). Th1 cells synthesize IL 2 and IFN to enhance acute GVH D, whereas Th2 cells prevent GVHD overall ( Pan et al., 1995). This profile shift, especially on transplantation donor cells, decreases the potential acute GVHD problem ( Zeng et al., 1997). Although there is a report regarding a decreased survival rate by immediate G CSF treatment after transplantation ( Ringdn et al., 2004), a number of other studies have demonstrated it to be a safe and effective treatment for transplantation ( Ho et al., 2003). G CSF has also been shown to have other clinical effects suc h as mobilization of CD34 + cells, neuroprotection and neurogenesis in the brain, angiogenesis, and anti apoptosis of cells expressing G CSFR ( Liongue et al., 2009). The mobilization ability of G CSF increases the amount of cells obtained from transplantat ion donors on both autogenic and allogenic transplantation ( Hart et al., 2009). Thus, the transplantation is able to be performed by using peripheral blood instead of bone marrow, so that the patient no longer needs to suffer from stem cell collection by t apping bone marrow. The overall outcome of the G CSF treatment for mobilization of blood collection and cost for the transplantation is more effective and superior than tapping bone marrow alone. One reason for CD34 + cell mobilization is that G CSF stimu lation induces their entry into an active G1 state from a resting G0 state ( Miles et al., 1990) EPO shares similar
20 mobilizing activity and this effect has been reported to be further enhanced when done in combination with G CSF treatment ( Marino and Rogu in 2008) On the other hand, the G CSF mobilization activity also affect s the s t imulation of other hematopoietic lineage cells such as red blood cells and platelets Combination therapy of G CSF and EPO is reported to stimulate production of more red blo od cell s than EPO treatment alone ( Miles et al., 1990) Also an increase in platelet counts is observed in patients treated with G CSF Following mobilization of stem cells by G CSF treatment other hematopoietic growth factors can then act upon the mob ilized stem cells thereby allowing for their differentiation. G CSF effect on neuronal precursor cells has been extensively studied by neuron research. G CSF is capable of travelling through the blood brain barrier and acts as a factor promoting neuroprot ection and neurogenesis ( Schneider et al., 2005 ; Lee et al, 2005 ). Neuronal precursor cells express G CSFR, and G CSF stimulation induces their differentiation within the brain, potentially preventing cortical cerebral ischemia Angiogenesis by G CSF tr eatment is a topic of interest in cerebral ischemia studies. Ischemia damage is reduced by the administration of G CSF and long term treatment aids in recovery via promotion of angiogenesis ( Meenhuis et al., 2009). Another important ability of G CSF is an ti apoptosis ( Demetri and Griffin 1991). An increase in total neutrophil counts is not only derived from the differentiation of early neutrophil precursor, but from the prolonged survival of mature neutrophils. T he enhancement of lifespan has also been reported in the other cells expressing G CSF receptor. In the report regarding the use of G CSF with anti thymocyte drug s for the prevention of type 1 diabetes, the authors focused on G as an anti
21 apoptosis aid for beta cells ( Parker et al., 2009). Moreover, G CSF supported the efficient skewing of regulatory T cell profile s from type 1 to type 2. 2.3. Intracellular Signaling from G CSF Receptor Ligation As more non hematopoietic abilities of G CSF are found, the intracellular signaling mech anism has become increasingly scrutinized. Among all of the signaling pathways discovered, JAK/STAT, PI3 K/PKB, and MAPK pathways are crucial for the proliferation and differentiation ability of G CSF stimulated cells ( Aaronson and Horvath 2002). The Janus Kinase (JAK) family consists of four tyrosine kinases: JAK1, JAK2, JAK3, and TYK2 ( von Vietinghoff and Ley 2008) Upon G CSFR ligation, JAK1 and JAK2 become activated and are critical for signal transduction ( Sherr and Roberts 1995) JAK signaling and activation promote receptor internalization and lysozomal degradation ( Bazan 1990). S ignaling also promotes further expression and cell surface levels of G CSFR. JAK1 is involved in the phosphorylation of the receptor and activation of Signal Transdu cers and Activators of Transcription (STAT) ( Sherr and Roberts 1995). The JAKs phosphorylate G CSFR creating a STAT docking site for their further activation by the JAKs, culminating in STAT nuclear translocation to mediate gene regulation ( van de Geijn et al, 2003). STAT1, 3, and 5 are involved in this signaling, however, only STAT3 and 5 are reported to play a role in the proliferation and survival signals of G CSF. STAT5 induces a weaker proliferation response than STAT3, however both STATs are inhib ited by suppressor of cytokine signaling (SOCS) 1 and 3 ( Zhuang et al., 2005). SOCS1 and 3 are negative regulators of JAK/STAT signaling and work to inhibit JAKs and STATs by targeting these components for degradation ( Zhuang et al., 2005 ; Yasukawa et al. 2000)
22 The proliferation response is also activated by Phosphatidylinositol 3 kinases (PI 3K)/protein kinase B (PKB) pathways which work to promote DNA synthesis ( Aaronson and Horvath 2002). MAPK and PI 3K/PKB pathways suppress Bid apoptotic signalin g, thereby further inhibiting apoptosis, while SH2 containing inositol phosphatase (SHIP) suppresses the PI 3K/PKB pathway ( Ward et al., 2000 ; Zhuang et al., 2005). 2.4. Interaction with Other Cytokines G CSF production is often regulated by the other cyto kines. Tumor necrosis factor (TNF) CSF (Koeffler et al., 1987 ; Koeffler et al., 1988). Sudden neutrophil increases observed in damaged tis sue areas predominantly involve TNF activity. Moreover, t issue damage leads to recruitment of macrophages where they are induced to express TNF CSF, further promoting macrophage activation. Intriguingly, TNF stimulation reduces cell surface expression of G CSFR on macrophages and has been shown to inhib it myeloid lineage differentiation (Elbaz et al., 1991). This inhibitory effect of TNF has furth er been demonstrated to inhibit growth of acute myeloid leukemia (Elbaz et al., 1991). The increase in the production of G CSF works to mobilizes stem cells in to blood circulation and induces their differentiation into granulocytes. Many more cytokines are involved in G CSF production. GM CSF and m ulti CSF increase G CSF production via promotion of monocytes differentiation and G CSF secretion (Oster et al., 1 989). Moreover, monocytes directly modify G CSF mRNA transcription (Ernst et al., 1989). IL family members also act as modu lators of G CSF production and IL 4 is known as a CSF inducer (Wieser et al., 1989). An increase in the number of macrophages at an inflammation site shows high release of IL 1, which enhances G CSF and GM CSF production from mesenchymal cells (Koeffler et al.,
23 1988). IL 1 especially stimulates the G CSF production from epithelial cells (Zsebo et al., 1988). On the other hand, G C SF also works to inhibit the inflammatory response and is reported to inhibit synthesis of inflammatory cytokines such as TNF 12, IL 1beta, and IFN ; Pan et al., 1995). Finally, IL 6, IL 8 and IL 10 are all reported to be synt hesized as G CSF concentration increases. These reports taken together suggest that G CSF has the ability to skew cytokine production toward an anti inflammatory response. Some cytokines share common non hematopoietic abilities and are therefore often use d in combination with G CSF. EPO is a growth factor that shares abilities and can wo rk synergistically with G CSF. Combination of G CSF and EPO treatment enhances red blood cell production of patients with low risk myelodysplastic syndromes by suppressio n of apoptosis of erythroid precursors (Liu et al., 2010). G CSF is reported to enhance hypoxia inducible factor 1 alpha and subsequent EPO production (Marino and Roguin, 2008). The combination therapy also showed high production of anti apoptotic factor s, neurotrophic factors, and stromal cell derived factor 1 for neuroprotection (Marino and Roguin, 2008). In addition, an increase in angiogenesis is observed in the combination therapy over either drug alone and is effective in ischemia (Marino and Rogui n, 2008). 2.5. HuG CSF and Species specific G CSF Patients who have a low amount of growth factors often develop hematological deficiencies (Gupta et al., 2010) This symptom is primarily caused by virus infections or chemo /radio therapy of cancer pati ents. Optimal treatment for these deficiencies has been well established by the availability of the recombinant growth factors in the
24 commercial market. These recombinants are designed with the natural sequence of each human growth factor and produced in expression vectors such as E.coli (Amgen Inc., 2004) Due to the fact that the growth f actor is naturally synthesized in the human body, application of these recombinants is generally well tolerated against any clinical adverse effects. HuG CSF (Filgras tim Amgen Inc., Tho usand Oaks, CA) is commercially available and is currently used as a symptomatic therapy in both humans and animals. Many clinical studies in humans have already shown a robust increase of neutrophils by daily prescribed treatments. Moreover, there is another commercial recombinant, Pegylated HuG CSF (PegHuG CSF: Pegfilgrastim Amgen Inc., Thousand Oaks) available by pegylating HuG CSF. As mentioned earlier, this pegylation decreases the frequency of the treatment by enhancing half life of G CSF. Application of the HuG CSF in animals shows similar efficacy and is recommended even in veterinary drug books. Commonly observed adverse effects in humans and animals are similar. A continuous use of the human sequenced drug in animals h as been reported to induce neutralizing antibodies against the drug (Fernndez Varn and Villamayor 2007, Arai et al., 2000 ; Hammond et al., 1991 ; Phillips et al., 2005 ; Randolph et al., 1999). In addition, a study regarding the use of HuG CSF in clinical ly normal dogs resulted in an induction of chronic neutropenia ( Phillips et al., 2005). The hematological disorder was caused by a state of G CSF deficiency mediated via production of neutralizing antibodies to endogenous CaG CSF which were produced in re sponse to exogenous HuG CSF ( Phillips et al., 2005). Thus, species specific growth factors, including FeG CSF or CaG CSF, have been developed experimentally and evaluated by researchers
25 to avoid this neutralizing phenomenon ( Hammond et al., 1991 ; Phillips et al., 2005 ; Randolph et al., 1999 ; Yamamoto et al., 2002 ; Yamamoto et al., 2009). 2.6. Pegylation Pegylation, polyethylene glycol (PEG) attachment to the therapeutic protein, extends half life of the drug and benefits commercial usage of the growth factor s in humans ( Molineux 2003, Curran and Goa 2002). Efficacy of a single injection of PegHuG CSF is comparable to that of twice daily injections of HuG CSF over a four day period. T he introduction of PegHuG CSF was shown to be beneficial for the comfort an d convenience of both the physician and the patients ( Lord et al., 2001). Therefore, the pegylated species specific growth factor could be a more relevant and suitable drug for clinical or commercial usage in veterinary medicine. T he large non immunogeni c PEG molecule can cover target epitopes of the protein thereby blocking potential immunogenic epitopes and preventing antibody mediated clearance In the present study, the hematological efficacy of PegFeG CSF was analyzed in FIV infected cats and uninfe cted cats and compared against unpegylated HuG CSF and FeG CSF in both short term and long term experiments. The aim of the study was to assess long lasting efficacy of PegFeG CSF in the short and long term studies and to detect neutralizing antibodies a gainst HuG CSF and possibly PegFeG CSF. As an alternative for unpegylated G CSF, PegHuG CSF has become a clinically relevant treatment for chemotherapy and/or transplantation In chemotherapy treated patients, a single dose of PegHuG CSF has replaced the daily, or even twice daily, injections of unpegylated G CSF. This may be because PegG CSF stimulated T cells show an unresponsiveness to alloantigen controlling T cell activity through IL 10 production, which eventually decreases GVHD likewise G CSF treat ment ( Morris et al.,
26 2004, Lindemann et al., 2005) Finally, compared to unpegylated G CSF, PegG CSF is reported to be a 10 fold more effective treatment against GVHD ( Rutella and Lemoli 2004). Thus, we sought to explore the use of PegFeG CSF over the un pegylated version.
27 CHAPTER 3 MATERIALS AND METHOD S 3.1. Animals A total of 21 cats were used in the treatment studies (Table 3 1). All cats were bred under specific pathogen free (SPF) condition at the Laboratory of Comparative Immunology and Retrovirolo gy (LCIR) animal facility at the University of Florida (Gainesville, FL) or purchased from Liberty Research, Inc. (Waverly, NY). Eight cats were vaccinated and protected against FIV challenge, while another eleven cats received either vaccination or place bo immunization and were unprotected after FIV challenge in a previous vaccine study in our laboratory. These 21 cats were transferred from the vaccine study, receiving no additional study treatments, for at least 4 months prior to the start of the FeG CS F study. Additional nine SPF cats were used as uninfected/untreated control group (not shown in Table 3 1). In the first cycle, cats that tested positive and cats that tested negative for FIV were each randomly assigned to treatment with PegFeG CSF, FeG CSF, or placebo consisting of PBS treatment. In treatment Cycles 2 5, all cats from the initial FeG CSF group were treated with PegFeG CSF and all cats from the first cycle PegFeG CSF group were treated with HuG CSF until they developed neutropenia (Table 3 1). Regardless of cycle, once the HuG CSF treated cats became neutropenic they were treated with PegFeG CSF. Additional seven cats (four FIV infected and three uninfected) were treated with HuG CSF for two cycles (Table 3 1) and those with HuG CSF ind uced neutropenia were not treated with PegFeG CSF to determine the neutrophil recovery rate in a SPF environment. All cats were monitored for clinical signs including infection or inflammation at the site of injection, ocular/nasal discharge, loss of
28 appe tite, abnormal behavior, and rectal temperature. Body weights were measured one day before each cycle of the study. 3.2. Production of FeG CSF and PegFeG CSF The cDNA encoding for mature feline G factor mating signal seq uence in pIC9K for secreted expression in yeast under control of the AOX1 promoter (Invitrogen, Carlsbad, CA). The recombinant Fe G CSF was collected by centrifugation, purified by ion exchange chromatography (High S Sepharose, Bio RAD Laboratories, Hercul es, CA), and was mono pegylated by reductive alkylation with sodium cyanoborohydride as previously described (Shi et al., 2007; Lee et al., 2008). Purity of the final product was analyzed by SDS PAGE with silver stain and by w estern blot analysis and was quantified in comparison to the appropriate dilution of HuG CSF ( Figure 3 1) Mouse m onoclonal antibody to FeG CSF was produced by Interdisciplinary Center for Biotechnology Research and was u sed in w estern blot analysis Biological activity was determin ed by NFS 60 based proliferation in comparison to the commercially available Hu G CSF (Filgrastim Amgen Inc., Thousand Oaks, CA). Both products were kept frozen until the study started. HuG CSF was purchased from the Veterinary Medicine Teaching Hospita l (VMTH) at the University of Florida and stored at 5C as recommended by the manufacturer. 3.3. Treatment Dose and Schedule The drugs used in this study were prepared to doses of 5, 10, 15 or 20 g/kg diluted in 1 mL PBS. The prepared dose was administe r ed subcutaneously in the dorsal neck of the animal. PegFeG CSF was administered once a week for the duration of the study with the exception for cat #VOD. Cat #VOD required additional treatments in a week during PegFeG CSF therapy of HuG CSF induced neu tropenia due to the
29 severity of the neutropenia. The schedule for HuG CSF treatment was once, 5 times, or 7 times a week depending on the cycle as described in the figure legend s. FeG CSF treatment in Cycle 1 was once a week. Each cycle consisted of 4 w eeks of drug treatment followed by 4 8 weeks of drug withdrawal (washout time) before the next cycle was initiated. 3.4. Hematological Values Blood collection was carefully scheduled based on the expected relative peak and trough of neutrophil count for each drug treatment. Initially, blood was collected on the fourth day after each treatment in Cycle 1 and also in the first and second weeks of Cycle 2. Collection was adjusted to the 16 20 hours post treatment thereafter based on finding from Cycles 1 a nd 2 as detailed in Result Section 3.5 Complete blood cell counts were performed on all samples. A Coulter A.C.T CBC instrument (Beckman Coulter) and Lasercyte (IDEXX Laboratories) were used with manual counts performed as routine confirmation of th e performance of the instruments. In addition, a control blood sample was routinely used to calibrate both instruments. Blood smears stained with Wright Giemsa were assessed for differential analysis by manual counting for all cycles. 3.5. Neutralizing Antibodies Sera from both HuG CSF treated cats and FeG CSF treated cats were tested for neutralizing activity against these drugs using a neutralization assay based on proliferation of recloned G CSF dependent NFS 60 cells (Phillips et al., 2005). Serial dilutions of test or control serum were mixed with equal volume of 0.05 ng HuG CSF followed by addition of 1.5x104 NFS 60 cells resulting in a total volume of 150 L in each 96 well. These samples were incubated for 5 days (37C, 5% CO2) prior to
30 addition of 20 L of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corp. Madison, WI) to measure the proliferation response using Molecular Devices Spectra MAX250 ELISA reader. Due to the limited supply of unpegylated FeG CSF, one time poin t in Figure 4 5 (Cycle 3) was also tested for neutralizing antibodies to FeG CSF. The drug neutralizing titers are shown as inverse of the dilution titer (1/x), whereby the dilution titer is the end point dilution that provides >50% inhibition of the prolif eration as compared to the standard negative control serum. 3.6. Pilot Study of PegFeG CSF Application in Dogs Four laboratory beagles purchased from Liberty were used in this study. All dogs were not castrated and kept in separate cages at animal fa cilities of the University of Florida. These dogs were divided in two groups and treated with either HuG CSF or PegFeG CSF. To compare the dose efficacy, the treatment dose and schedule was kept same for both drugs. The treatment dose was 15 g/kg once a week and was injected subcutaneously at dorsal neck. Any clinical signs were monitored. 3.7. Statistical A nalysis The results from Fe GCSF, PegFeG CSF and HuG CSF treatment groups were tested for normality by Shapiro Wilk followed by T test as parametr ic analysis for population with normal distribution and nonparametric two way analysis of variance (ANOVA) for population without normal distribution. The comparison among three or more groups were made using ANOVA with Bonferroni correction. Likewise, c omparison between pre vs. post treatment neutrophil values for FeG CSF, PegFeG CSF, and HuG CSF groups was tested for normality followed by either n onparametric ANOVA or parametric paired T test. The statistical analysis to compare relative risk for deve loping neutropenia between drug treatments or between FIV infected and
31 uninfected groups was determined by using Fisher Exact test for non numerical values. Key s tatistic al results not discussed in the text are also listed in Table 3 2 and a com parison was considered to have statistically significant difference when p <0.05. The above statistical analyses were performed using SigmaPlot for Windows version 11.0 (Systat Software, Inc., San Jose, CA).
32 Table 3 1 Brief d escr iption of a nimals u sed in t his s tudy ID Sex Age a FIV Status b Treatments c Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 GW4 F 1 year Uninfected/Challenged Fe Peg Peg Peg Peg H9N M 1 year Uninfected/Challenged Fe Peg Peg Peg Peg JV3 F 1 year Uninfected/Challenged Fe Peg Peg Peg Peg H9K M 1 year Uninfected/Challenged Peg Hu Hu/Peg Peg Peg H9O F 1 year Uninfected/Challenged Peg Hu Hu/Peg Peg N/A VOD F 1.5 years Uninfected/Challenged Peg Hu Hu/Peg Peg Peg GY1 F 1 year Infected Fe Pe g Peg Peg N/A H9L M 1 year Infected Fe Peg Peg Peg N/A H9P F 1 year Infected Fe Peg Peg Peg N/A H5O M 1 year Infected Peg Hu Hu Hu N/A H9M M 1 year Infected Peg Hu Hu/Peg Peg N/A JV2 F 1 year Infected Peg Hu Hu Hu N/A GX2 M 1 year Infected control co ntrol control N/A N/A GW2 F 1 year Uninfected/Challenged control control control N/A N/A H9W M 6 months Uninfected/Unchallenged Hu Hu/Peg N/A N/A N/A H5Y F 6 months Uninfected/Unchallenged Hu Hu N/A N/A N/A VVE M 1.5 years Uninfected/Challenged Hu Hu N /A N/A N/A H9U F 6 months Infected Hu Hu N/A N/A N/A CX3 F 1 year Infected Hu Hu N/A N/A N/A CV6 F 1 year Infected Hu Hu N/A N/A N/A CU3 M 1 year Infected Hu Hu N/A N/A N/A a Age at the initiation of the study b Vaccinated cats after challenge that are protected are shown as Uninfected/Challenged, while unchallenged cats are shown as Uninfected/Unchallenged. c Abbreviations are PegFeG CSF (peg), unpegylated FeG CSF (Fe), HuG CSF (Hu), PBS (control), and not applicable (N/A).
33 Table 3 2. Statistical a nalysis Groups Compared a Number of Cats P value b PegFeG CSF vs. FeG CSF (Cycle 1) Mean high peak (Day 4) 12 0.002* Mean low peak (Day 7) 12 <0.001* c FIV positive 6 0.038* FIV negative 6 0.003* PegFeG CSF vs. HuG CSF (Cycle 2) High peak (16 20 hours) 12 0.018* Therapeutic PegFeG CSF treatment (Cycle 2) Post treatment peak response (16 20 hours) 5 0.039* Pre vs. Post treatment Peak PegFeG CSF (Day 4) 6 <0.001* FeG CSF (Day 4) 6 0.033* c HuG CSF (16 20 hour s) 14 <0.001* c FIV effect on treatments 12 0.17 Risk of neutropenia HuG CSF vs. PegFeG CSF 19 0.011* c Uninfected vs. Infected 0.07 c a Time of blood collection after each treatment shown as (16 20 hours, Day 4, or Day 7). b Statistically sig nificant (*) based on p <0.05. c P value of statistical comparisons not described in the text.
34 Figure 3 1. SDS PAGE with s ilver stain of the PegFeG CSF preparation used in the animal studies Purity of the final product wa s determined by silver st ain analysis of the SDS PAGE Different concentrations of PegFeG CSF (79,000 dalton) and commercial HuG CSF (19,000 dalton) w ere applied to a 12% polyacrylamide gel. The protein bands were visualized by Silver Stain Plus (Bio Rad Laboratories, Inc Her cules, CA) The lane designation is shown as (a) molecular weight standards; (b) PegFeG CSF ( 66.5 ng); (c) PegFeG CSF (133 ng); (d) HuG CSF (180 ng) ; (e) HuG CSF (225 ng). Western blot analysis (data not shown) confirmed that monoclonal antibody to FeG C SF react ed strongly to the 79 ,000 dalton band when compared to the reactivity of the double bands around 19 ,000 dalton The band less than 10,000 dalton in lane c was negative for FeG CSF by Western blot. Thus, the PegFeG CSF preparation used in cats con tain s high level of PegFeG CSF with residual unpegylated FeG CSF
35 CHAPTER 4 RESULTS 4.1. Comparison of N eutrophil P roduction between FeG CSF and PegFeG CSF All of the neutrophil counts of cats treated with FeG CSF and PegFeG CSF dramatically increased following the third treatment ( Figure 4 1). The cats treated with PegFeG CSF showed a more consistent and robust mean increase in neutrophil count than with FeG CSF group at Day 18; Tab le 3 2 p =0.002). Nevertheless, weekly treatment of FeG CSF provided significant increase in mean neutrophil counts when compared to pre treatment values by Day 18 ( p =0.017). Consistent with previous studies from our laboratory (Phillips et al., 2005), un infected cats responded to the treatment with higher mean neutrophil counts than the FIV vs 4.2. Major Difference in Neutrophil P roduction between PegFeG CS F and HuG CSF In the study cycles comparing once weekly dosing with PegFeG CSF and daily HuG CSF, the weekly PegFeG CSF produced higher neutrophil counts than the daily HuG G CSF group vs. 50,477 neu CSF group in Cycle 2; p= 0.018) ( Figure 4 2A). Robust total neutrophil counts were observed throughout Cycles 2 5 for PegFeG CSF ( Figure s 4 2A and 4 2B) along with an increase in band neutrophils indicating left shift ( Figure 4 3). More importantly, an additional two treatments in Cycle 5 at Weeks 66 and 67 post first FeG CSF treatment markedly increased the neutrophil counts of the uninfected cats ( Figure 4 2B). Thus, these cats even over one year of treatment with a total cumulative
36 P egFeG CSF dose of 175 g/kg or cumulative FeG CSF/PegFeG CSF dose of 200 g/kg did not develop neutralizing antibodies to PegF G CSF ( Figure 4 2B). Since not all cycles had 4 weeks of treatment, the first two treatments of Cycles 2 5 are shown along with t hree treatments of Cycle 1 to demonstrate the consistent increase in neutrophil counts throughout Cycles 3 5 ( Figure 4 2B). No statistical difference was observed among the mean values of the second treatment in Cycles 3 5 for the uninfected group ( p= 0.56 6). The neutrophil values of Cycle 2 (blood collection on Day 4 post treatment for the first two treatments) were lower than those of Cycles 3 5 (blood collection at 16 20 hours post treatment) presumably due to the difference in the blood collection time ( Figure 4 2B). The peak neutrophil values were detected 16 20 hours post treatment (see section 4 .4). Moreover, the highest neutrophil numbers were detected after the third or fourth PegG CSF treatments of the given cycle ( Figure 4 2A). Generally, the neutrophil peaks within a cycle trend lower in the FIV infected cats than the uninfected cats as shown for the first and second treatments of Cycle 4 ( Figure 4 2B), although a statistical difference was not observed (first treatment, p= 0.175; second treatm ent, p= 0.177). In addition, the infected group seemed to have substantially lower neutrophil counts than the uninfected group at the third and fourth treatments of Cycles 2 and 3 (data not shown), but the difference was not statistically significant ( p> 0. 08) likely due to the small sample size. In contrast to the FeG CSF and PegFeG CSF induced neutrophil increases, 4 of 6 HuG CSF treated cats developed neutropenia in the first two cycles of HuG CSF treatments ( Figure 4 2A, Cycles 2 and 3). HuG CSF tr eated cats with rapid neutropenia and those responsive to HuG CSF for three cycles were further compared ( Figure 4 2A,
37 dashed lines). Cats with rapid neutropenia showed a decline in neutrophil counts after the third or fourth treatment of first HuG CSF trea tment cycle ( Figure 4 2A, Cycle 2), which continued until after the second HuG CSF treatment cycle ( Figure 4 2A, Cycle 3). The total cumulative HuG CSF dose received by the cats at onset of the neutropenia (neutrophil count of <2500) was 225 235 g/kg. C ats responsive to HuG CSF had peak neutrophil values that became lower with each additional cycle ( Figure 4 2A) but did not develop neutropenia even after the third treatment cycle with HuG CSF ( Figure 4 2A, Cycle 4). 4.3. Additional HuG CSF S tudy to D eter mine the D uration of HuG CSF I nduced N eutropenia In the second HuG CSF study, three 6 months old cats ( n=1 infected; n=2 uninfected) along with four 1 to 1.5 years old cats (n= 3 infected; n= 1 uninfected) received HuG CSF treatments with a dose (5 g/kg at 7X per wk) as per the recommended clinical dose (Plumb, 2008) in the first cycle and doses of 5 15 g/kg in the second cycle ( Figure 4 4). One of the three young cats ( uninfected #H9W) started to decline in neutrophil counts on or after the third treatmen t of Cycle 1 and developed severe neutropenia during the washout time while an other y oung cat ( uninfected #H5Y) and a n old er uninfected cat (#VVE) developed neutropenia after the third treatment of Cycle 2 at about the same time as the two infected cats ( young cat #CV6 and older cat #H9U) ( Figure 4 4). The c umulative HuG CSF dose for the two young cats (#H9W and #H5Y) was 185 g/kg each. Cat #H9W was transferred to PegFeG CSF therapy after 3 week s of the washout as described in section 4 .7 while the rem aining four neutropenic cats were monitored during washout period (4 8 weeks) and some beyond for the time required to recover neutrophil counts to 2500 Both the recovery rate and severity of
38 neutropenia were compared to the neutrophil counts of the two HuG CSF responsive cats (#CX3 and #CU3) at the same time points Following the two treatment cycles, one uninfected cat (#H9W) remained neutropeni c until 1.5 weeks into the washout period, while another uninfected cat (#VVE) and one infected cat (#H9U) remained neutropenic until 3 weeks of washout ( Figure 4 4). One uninfected cat (#H5Y) and one infected cat (#CV6) had gradual increases in neutrophi l counts to 2,500 and 3,000 neutrophils/L, respectively after 3 weeks of washout. These results suggest that the duration of neutropenia during washout period were at least 1.5 3 weeks for the majority of HuG CSF induced neutropenic cats. One of the two HuG CSF responsive infected cats (#CU3) had transient neutropenia at third week of the washout period, but recovered by 3 months of no treatment, while the other responsive cat (#CX3) had normal neutrophil levels throughout the 5 months tested without tre atment. Similar to the first study, FIV infected cats were more resistant to developing HuG CSF induced neutropenia. In summary (sections 4.2 and 4 .3), all six uninfected cats from HuG CSF Studies 1 and 2 developed neutropenia b efore or during the secon d cycle, while 3 of 7 FIV infected cats developed neutropenia during or after the second cycle ( Figure s 4 2A and 4 4). 4.4. Effect of Age and FIV S tatus on N eutrophil L evels of HuG CSF or PegFeG CSF Treated C ats A notable observation of HuG CSF Study 2 was the low neutrophil counts in all cats after HuG CSF treatment ( Figure 4 4). Although group numbers were very small, given that two different age groups and two groups with or without FIV infection were used, these factors could have contributed to the low neutrophil levels after the full
39 course of treatment or even during the washout period of the later cycles. T o assess the overall effect of these factors, n eutrophil values at pre and post treatment at the last week of washout period or after 1 2 months of no treatment were compared to those of age matched / uninfected control group ( Figure 4 6 ) No significant difference was observed in the mean neutrophil values: 1) between the same age groups of the control group and the individual treatment group, 2) between pre and post treatment groups from the same treatment or even between the two treatment groups, and 3) among all three age groups of the control. Thus, the age of the cats had no significant effect on the neutrophil values with or without treatm ent. Although not statistically significant, general trends observed were ( Figure 4 6 ): 1) the higher mean neutrophil values at all ages (0.5, 1, and 1.5 years) of the uninfected group compared to the FIV infected groups with exception of post HuG CSF g roup at 1.5 years of age, 2) a substantial decrease in neutrophil counts from the pre treatment group at 6 months of age to the post HuG CSF treatment group at 1 year of age, 3) a substantial increase in neutrophil counts after the treatment with PegFeG CS F, and 4) a decreasing trend in the mean neutrophil values after treatment with HuG CSF. 4.5. Determining the O ptimal Blood C ollection S chedule The treatment and blood collection schedule of the cats was carefully evaluated and modified for more accurate r esults over the course of the study. Major differences in neutrophil counts were observed when blood collection was changed to 16 20 hours vs. the fourth day after each PegFeG CSF treatment in Cycle 2 ( Figure 4 2A, Day 4 post treatment for first two weeks vs. 16 20 hours post treatment for last two weeks). Also, compared to blood samples collected on the fourth or seventh day after treatments (both done in Cycles 1), blood samples collected 16 20 hours post treatment
40 (Cycles 2 5) displayed higher percenta ges and absolute numbers of band neutrophils indicating more regeneration (data not shown). Occasionally, neutrophil counts for some cats peaked at 36 hours post treatment (data not shown). This study demonstrates that the peak neutrophil counts can be d etected by 24 hours post treatment even with FIV infected cats and should be the time point tested for clinic animals undergoing HuG CSF therapy. 4.6. Neutralizing A ntibodies to HuG CSF and PegFeG CSF The severity of neutropenia ( Figure 4 4) or resistance to PegFeG CSF therapy ( Figure 4 5, see section 3.8) correlated with higher neutralizing antibody titers to HuG CSF in multiple cats and a modest titer to FeG CSF in one cat (uninfected cat #VOD). HuG CSF induced neutropenia occurred in 6 of 6 uninfected cat s, while only 3 of 7 infected cats developed neutropenia ( Figure s 4 2A and 4 4). This observation suggests that cross reactive neutralizing antibody titers to the endogenous FeG CSF may be lower in the infected cats than in the uninfected cats, even though the mean neutralizing antibody titer to HuG CSF of the infected cats (1286 titer/cat) was slightly higher than that of the uninfected cats (1100 titer/cat). In contrast, none of the PegFeG CSF treated cats developed neutralizing antibodies to PegFeG CSF or HuG CSF ( Figure s 4 2A and 4 2B). 4.7. Other H ematological V alues, C linical S igns, and FIV L oad Total white blood cell (WBC) values were similar to the production profile of neutrophils throughout the course of study, whereas lymphocyte, eosinophil, mono cyte, and basophil counts did not change (data not shown). Relatively higher eosinophil and monocyte counts were observed in Cycle 4. However, these values were higher prior to the initiation of the cycle. Other hematological factors including RBC, hemo globin,
41 packed cell volume, total protein, and platelets did not change over the course of study. Regardless of treatment group, none of the cats showed any signs of infection, irritation, or inflammation at the site of injection. Unlike the adverse effe cts (fever and diarrhea) observed in human patients receiving HuG illnesses or abnormalities were observed in either the PegFeG CSF or the HuG CSF treated cats with the exception of the hematological abnormali ty, neutropenia in HuG CSF treated cats. Most likely the SPF environment of the cat housing prevented secondary infections which are often seen in neutropenic cats. FIV titration of all cats used in the study (determined during the washout period after C ycles 2 or 3) showed no increase in FIV titers when compared to the FIV titers detected before any G CSF treatment (data not shown). 4.8. PegFeG CSF Therapy of HuG CSF I nduced N eutropenia In the evaluation of therapeutic effects of PegFeG CSF, one FIV infe cted and four uninfected cats with HuG CSF induced neutropenia, transferred from Cycles 2 or 3 of HuG CSF studies ( Figure s 4 2A and 4 4), were treated with PegFeG CSF ( Figure 4 5, data not shown for #H9W). Three of 4 cats (#H9K, #H9M, and #H9O) responded immediately to the first treatment (8,829, 12,342, and 12,384 neutrophils/ L, respectively) and two of them had a concurrent decline in neutralizing antibody titer to HuG CSF in Cycle 3. The remaining cat (#VOD) with the most severe neutropenia required additional dosing and responded modestly in Cycle 3 (7,992 neutrophils/L) and more substantially in Cycle 4 (17,282 40,858 neutrophils/L). The high neutrophil increases coincided with the time when neutralizing antibody titers became undetectable even a fter receiving additional doses of PegFeG CSF. The PegFeG CSF dose needed to recover from neutropenia was a single dose of 15 g/kg for the three
42 cats (#H9K, #H9O and #H9M) and a cumulative 110 g/kg for the severely neutropenic cat (#VOD). In another se t of cats ( Figure 4 4), only one cat (#H9W) was treated PegFeG CSF due to availability of the product and it responded finally with three treatments (cumulative dose of 45 g/kg, data not shown). Statistically significant therapeutic efficacy was observed with PegFeG CSF therapy of HuG CSF induced neutropenia ( p= 0.039) (Table 3 1). 4. 9 Inter species U se of PegFeG CSF A preliminary study on hematopoietic efficacy of PegFeG CSF treatment in dogs was performed to determine whether the drug can be used in dog s. The high amino acid (aa) sequence identity of over 90% between cats and dogs ( Figure 4 7) suggests that PegFe GCSF should be effective in dogs. Due to the limited supply of PegFeG CSF after feline studies, two laboratory dogs were treated PegFeG CSF o nce a week for two weeks at 15 g/kg, while two heavier dogs were similarly treated with HuG CSF ( Figure 4 8). Both drugs induced high levels of neutrophils. The heaviest dog (#D07) with the highest cumulative dose had the highest increase in neutrophil l evels in the two week treatment. In the cat studies, cats t reated with higher cumulative PegFe G CSF dose trended to have higher neutrophil levels (data not shown). Similar effect was also observed in HuG CSF (Amgen Inc. 2004 ) I n the cat studies, two we eks of treatment with HuG CSF did not cause adverse effects such as the production of neutralizing antibodies to the drug or to the endogenous G CSF. Similarly in this short treatment study in dogs, no significant difference was observed in the level of n eutrophil increases between PegFeG CSF group and HuG CSF group. Reported studies demonstrate longer treatment with HuG CSF will cause a loss of drug activity in many dogs and subsequent neutropenia in a
43 portion of them ( Schuening et al. 1989) Sequence analyses revealed that aa identity between humans and dogs and between human and cats is about 80% ( Figure 4 7). This analysis suggests that there are as much potential epitopes on HuG CSF that can drive neutralizing antibody production in dogs similar to those observed in cats ( Figure 4 4). Moreover, the >90% sequence identity between FeG CSF and CaG CSF may suggest less potential for PegFeG CSF to induce anti drug neutralizing antibodies in dogs than HuG CSF. However, longer treatment studies will be n eeded to determine the potency and safety of using PegFeG CSF in dogs.
44 Figure 4 1. Neutrophil numbers in cats treated with FeG CSF (Fe) or PegFeG CSF (Peg) in Cycle 1. Six FIV infected cats and six uninfected cats were treated once a week with FeG C SF (Fe, n=3 infected and n=3 uninfected), PegFeG CSF (Peg, n =3 infected and n=3 uninfected), or PBS (control, one infected and one uninfected) for a total of 3 weeks. The treatment time and dose are shown with gray and black boxes and arrows. The dose fo r both drugs was 5 g/kg on the first week and 10 g/kg on the second and third weeks. The average results from the blood samples collected on the 4th day after each treatment are shown with SD, except for the control group. Significant differences were observed between the combined means of PegFeG CSF vs FeG CSF groups ( p =0.002) (***) and between the individual means of PegFeG CSF FIV negative vs. PegFeG CSF FIV negative groups ( p =0.003) (**) and PegFeG CSF FIV positive vs. FeG CSF FIV positive groups ( p =0.038) (*) ( Table 3 2 ). The normal reference range used by the Veterinary Medical Teaching Hospital at the University of Florida is shown as a grey zone (neutrophil values of 2,500 to 12,500 cells/L).
45 Figure 4 2. Decreased numbers of neutr ophils due to anti drug neutralizing antibodies and activities were seen in cats treated with HuG CSF (Panel A) but not in PegFeG CSF treated cats (Panels A and B). The mean neutrophil count with SD is shown for the time points tested and cats were consi dered neutropenic at <2500 neutrophils/L which is shown as below the lower limit of the normal reference range (grey zone) (A and B). Neutralizing antibody titers to HuG CSF are shown below the figure along with treatment dose, frequency, and cumulative d ose under each cycle (A and B). The neutralizing antibody value of < 300 is designated as undetected (UD). All cats were given a washout period (light grey line s ) of 4 8 weeks between the treatment cycles (A and B) (A) HuG CSF or PegFeG CSF treatment of animals continued from Figure 4 1 comprising Cycles 2 4. FIV infected (n=3) and uninfected (n=3) cats (combined mean neutrophil numbers) were treated 5 times per week (5X/wk) with HuG CSF (Hu) at 10 g/kg in Cycle 2, 7X/wk at 2 5 g/kg in Cycle 3 with recom mended clinical doses (Plumb, 2008), and 1X/wk at 15 g/kg in Cycle 4. Additional FIV infected (n=3) and uninfected (n=3) cats (combined mean neutrophil numbers) were treated with PegFeG CSF (P) 1X/wk at 15 g/kg in Cycles 2 4 except for the first treatme nt in Cycle 2 at 10 g/kg (four treatments for Cycles 2 and 3; two treatments for Cycle 4). The treatment was withdrawn from four cats (nonresponders, NR) on Day 75 (#VOD and #H9M) and on Day 77 (#H9K and #H9O) corresponding to the first week of Cycle 3. Only two infected cats remained responsive to HuG CSF treatments (responders, R) and received all three treatment cycles with HuG CSF.
46 (B) An additional fifth treatment cycle with PegFeG CSF shown with treatment Cycles 1 4 from Figures 4 1 and 4 2A and a s a comparison of FIV infected cats (n=3, open symbol) and uninfected cats (n=3, closed symbol) from panel A. These cats received FeG CSF in Cycle 1 ( Figure 4 1) and were treated 3 4 more cycles with PegFeG CSF (P, solid lines found in Figure 4 2A). During C ycle 5 the cats were treated with PegFeG CSF (P) 1X/wk at 15 g/kg for only 2 weeks like Cycle 4. Only the first two week results are shown for Cycles 1 3 to prevent miss interpretation of the data by synchronizing the data with those of Cycles 4 and 5 as there were only 2 treatments in each of these cycles. Blood collection was performed on the fourth day following treatment in Cycle 1 (a,b) and for the first two weeks of Cycle 2 (c,d), and 16 20 hours following treatment in the remaining cycles.
47 Figure 4 3. Comparison of hematological blood smears from pre and post treatment samples. Blood smears from cat #H9N that responded robustly to the PegFeG CSF treatments are shown. The pre treatment smear was collected when the neutrophil value was 5,0 84 cells/L, and the post treatment smear was collected when the neutrophil value was 66,521 cells/L. Increased number of band neutrophil was observed in the post treatment smear. Thus, PegFeG CSF treatment caused left shift to more immature neutrophils in the peripheral blood.
48 Figure 4 4 Additional HuG CSF study to characterize HuG CSF induced neutropenia. Previously untreated FIV infected cats (n=4, black solid lines) and uninfected cats (n=3, grey dashed lines) were treated daily for 4 weeks wi th the recommended treatment dose of 5 g/kg HuG CSF (Plumb, 2008) during Cycle 1 After 6 weeks of washout period (light grey lines), two cats (#H9W and #H5Y) received 1X/wk of 15 g/kg dose for 3 weeks in Cycle 2, while remaining cats received daily (7X /wk) treatment of 5 g/kg dose for the first two weeks and 10 g/kg dose for the following 2 weeks. After Cycle 2, two cats (#H9W and #H5Y) and five cats were evaluated for neutrophil level after washout period of 1.5 weeks and 3 weeks, respectively at ti me point ( a ). Subsequently, one cat (#CU3) and five remaining cats except for cat #H9U were tested at 3 months and 5 months of no treatment, respectively, shown as time point ( b ). All cats at pre treatment time were negative for neutralizing antibodies t o HuG CSF (undetectable, UD). The normal neutrophil range is shown as light grey zone.
49 Figure 4 5. PegFeG CSF therapy of cats with HuG CSF induced neutropenia. One FIV infected (#H9M) and three uninfected cats (#H9K, #VOD, and #H9O) from Figure 4 2A d eveloped neutropenia by HuG CSF treatment (Hu, dashed lines) and were transferred to the therapeutic study with PegFeG CSF (Peg, solid lines). Except for cat #VOD, all neutropenic cats were treated with 15 g/kg of PegFeG CSF 1X/wk for 3 weeks in Cycle 3 and for 2 weeks in Cycle 4 along with a washout period (light grey lines) of 6 8 weeks between cycles. Cat #VOD with the most severe neutropenia required additional dosing at 20 g/kg on Week 1 (open black arrow with a ) of Cycle 2 and finally responded to PegFeG CSF after another 15 /kg dose of PegFeG CSF on Week 12 (open black arrow with b ) of Cycle 3. Neutralizing antibody level against HuG CSF is shown below each cycle, while those also against FeG CSF are shown below Cycle 3 next to HuG CSF as (HuG C SF/FeG CSF). The neutralizing antibody value of <300 is considered undetectable (UD) as shown in Cycle 4 for cat #VOD. The normal reference range for neutrophil is shown as light grey zone.
50 Figure 4 6 Effect of age and FIV infection on neutrophil levels among cats treated with HuG CSF or PegFeG CSF. Neutrophil values at pre and post treatment were compared to those of age matched uninfected control group. The post treatment values for cats with HuG CSF treatment were obtained at 1 or 2 months a fter the completion of treatment for the cats in HuG CSF Study 2 or at the last week of washout period closest to the age shown for the cats in HuG CSF Study 1. The post treatment values for cats with PegFeG CSF were also obtained at the last week of wash out period closest to the age shown. The neutrophil count of FIV infected cats (open circle with dashed line for mean value) and uninfected cats (closed black circle with solid line for mean value) are shown in comparison to their corresponding ages (0.5, 1, or 1.5 years of age). All cats from the PegFeG CSF or HuG CSF studies were included with the exception of the two HuG CSF treated cats (#VOD and #VVE) with the pre treatment age of 1.5 years. Neutralizing antibodies to HuG CSF ranging in titers from 3 00 to 4800 developed in all cats receiving HuG CSF therapy ( Figure s 4 2A, 4 4, and 4 5). Neutropenia induced by anti HuG CSF neutralizing antibodies occurred in cats with titers as low as 600 ( Figure 4 5). Cat #VOD that had the slowest response to rescue treatmen t with PegFeG CSF had the highest titer to HuG CSF of 4 800 and a titer to FeG CSF of 300 in Cycle 3 ( Figure 4 5). So far no other cats in Cycle 3 ( Figure 4 5) had detectable neutralizing titers to FeG CSF, but earlier cycles at peak anti HuG CSF neutralizing titers still need to be analyzed.
51 Figure 4 7 Comparison of G CSF sequences between Hu Fe and Ca G CSF.
52 Figure 4 8. Inter species u se of PegFeG CSF. Laboratory beagles receiving either PegFeG CSF (n=2) or HuG CSF (n=2) were evaluated f or G CSF responses by monitoring their neutrophil counts. These results were from the blood samples collected 24 hr after each treatment. The reference range is shown as a gray zone between maximum (11 500 counts) and minimum (3 000 counts) limits fo r ne utrophils in dogs ( Hematology Normal Values reference for UF VMTH ). The cumulativ e dose is presented below the identification number for each dog. Due to the limited supply of PegFeG CSF after the feline studies, two laboratory dogs with the lowest weigh t were treated with PegFeG CSF at 15g/kg once a w ee k for 2 w ee k, while two additional laboratory dogs were similarly treated with HuG CSF. Monitoring of the samples was performed one day after the treatment.
53 CHAPTER 5 DISCUSSION PegFeG CSF appears to be a superior treatment for neutropenia in feline patients. Short and long term treatments with PegFeG CSF provided therapeutic and sustainable neutrophil stimulation without the development of neutralizing antibodies in both uninfected and FIV infect ed healthy cats. This suggests that a safe, long term PegFeG CSF treatment of neutropenia from chronic disease is possible without the development of potentially fatal hematopoietic dyscrasias. Weekly doses of PegFeG CSF induced higher peak neutrophil pro duction and showed greater sustained activity than weekly treatment with a similar dose of FeG CSF ( p =0.002) or daily HuG CSF treatments ( p =0.018). PegFeG CSF provided the most therapeutic and sustainable neutrophil production ( p <0.001) in both uninfected and FIV infected cats without the development of neutralizing antibodies to HuG CSF, suggesting the presence of cross reactive antibodies to endogenous G CSF in a majority of the cases with severe neutropenia. PegFeG CSF also was effective in rescuing an imals with HuG CSF induced neutropenia, resulting in return of clinically normal neutrophil numbers ( p =0.039). Hematopoietic disturbances are a major clinical complication in certain infectious diseases including FIV infection, bone marrow or stem cell tra nsplantation, or cancer therapy (Brown and Rogers, 2001, Calhoun et al., 2005; Fleming et al., 1991; Linenberger et al., 1991; Lothrop et al., 1988; Roy Ghanta and Orange, 2010; Schuening et al., 1989; Yanay et al., 2006). Commercially available recombinan t HuG CSF has been used for enhancing neutrophil production in animals with neutropenia (Calhoun et al., 2005; Lothrop et al., 1988; Roy Ghanta and Orange, 2010; Schuening
54 et al., 1989; Yanay et al., 2006). However, neutralizing antibodies against HuG CSF have resulted in chronic neutropenia in dogs and cats (Hammond et al., 1991; Plumb, 2008). In our study, 69% (9 of 13) of cats treated with HuG CSF developed neutropenia by Cycle 2 (cumulative 12 weeks), suggesting the development of neutralizing antibod ies against HuG CSF that also cross reacted with endogenous G CSF and affected neutrophil production. This view is substantiated by the detection of neutralizing antibodies to HuG CSF in all HuG CSF treated cats in current studies ( Figure s 4 2A, 4 4, and 4 5) and the higher neutralizing antibodies (4 800 and 1 200) observed in the neutropenic cats more resistant to PegG CSF rescue therapy ( Figure 4 5). Thus, findings from current and other recent studies raise concern over the use of human derived drugs in veterinary therapy (MacLeod et al., 1998; Phillips et al., 2005; Randolph et al., 1999; Randolph et al., 2004). Our study showed short and long term treatments with PegFeG CSF provided therapeutic and sustainable neutrophil stimulation without the deve lopment of neutralizing antibodies in both uninfected and FIV infected healthy cats. This suggests that a safe, long term PegFeG CSF treatment of neutropenia from chronic disease is possible without the development of potentially fatal hematopoietic dyscr asias. The development of neutralizing antibodies to HuG CSF appears to correlate with cumulative dose. No neutralizing antibodies against PegFeG CSF were detected either in neutralizing antibody analysis or based on response to therapy. However, due to the high efficacy of PegFeG CSF, cumulative doses comparative to HuG CSF were only achieved after Cycle 5 for the uninfected cats with cumulative PegFeG CSF dose of 175 g/kg or cumulative FeG CSF/PegFeG CSF dose of 200 g/kg. Given that the
55 cumulative d oses of HuG CSF required to initiate neutropenia were 225 235 g/kg in first study and 185 g/kg in second study, the cumulative FeG CSF/PegFeG CSF dose of 200 g/kg approaches comparable amounts that caused HuG CSF induced neutropenia. In the portion of t he study looking at PegFeG CSF as a viable treatment for HuG CSF induced neutropenia, two cats were delayed in response to treatment. The presence of cross neutralizing antibodies between HuG CSF and FeG CSF most likely caused the delay suggesting that suc h antibodies would need to be cleared or suppressed prior to or during effective treatment. In addition, our comparative study of FIV infected and uninfected cats treated with HuG CSF showed that more than half of the FIV infected cats (4 of 7) did not de velop neutropenia while all of uninfected cats did ( Figure s 4 2A and 4 4). Although a mechanism is not definitely determined, it is hypothesized in current study that the resistance to neutropenia in FIV infected cats is most likely that the immunosuppress ion, which is commonly seen in FIV infected animals (Yamamoto et al., 2007), potentially affecting the production of anti HuG CSF neutralizing antibodies. Further investigation combining the use of immunosuppressive therapy, such as cyclosporine and predn isolone, in conjunction with PegFeG CSF may neutralizing antibodies (Sawada et al., 2008). The preliminary study of PegFeG CSF use in laboratory dogs demonstrated that PegFeG CS F can enhance neutrophil numbers even in dogs. The dogs treated with HuG CSF had relatively higher neutrophil numbers than PegFeG CSF, however, this study is somewhat skewed by the difference in weight of the animals. As described in
56 the PDR for HuG CSF (Neupogen ), total dose dependent activity of the G CSF may affect the results as observed in the cat studies (Note that the cats but not the dogs were evenly distributed to the treatment groups based on their weight to eliminate total d ose dependent eff ect of G CSF.) Due to availability of the drug, the smallest dogs are distributed in PegFeG CSF treatment group, which may have caused appearance of relatively lower efficacy. Additional studies will be needed to determine the potency and safety of using PegFeG CSF in dogs. A major concern in veterinary medicine is client compliance often influenced by the frequency and mode of treatment. Pegylation of HuG CSF has already been shown to increase half life and decrease serum clearance (Curran and Goa, 2002 ; Molineux, 2003; Kobbe et al., 2009). In fact, several comparative studies report single weekly treatments of PegHuG CSF are equivalent to that of daily injections of unpegylated HuG CSF (Castagna et al., 2010; Lord et al., 2001). Similarly, our results suggest pegylation of species specific growth factors increases the half life and decreases serum clearance. Because of the higher and more sustained response, PegFeG CSF provides the clinician with more flexible treatment protocols. Weekly subcutaneous i njections can be administered on an outpatient basis or at home by the owner, improving patient comfort and significantly increasing compliance.
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67 BIOGRAPHICAL SKETCH Yohichi Sakagawa was born in Nagano, Japan, to Takashi and Chihi ro Sakagawa He resided in the large urban town of Tokyo, Japan, for 18 years. In 2005, he graduated high school and decided to study abroad at the University of North Alabama In August of 2007, he transferred to the University of Florida to where he received a Bachelor of Science degree in Ani mal Science. He was accepted into the graduate program at the College of Veterinary Medicine at the University of Florida in Fall of 2009. In December of 2011, he will receiv e h is Master of Science degree in v eterinary medical s cience s a nd then p ursue a Ph.D. degree.